Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1

Meinander, Nina Q.; Boels, Ingeborg C.; Hahn‐Hägerdal, Bärbel

doi:10.1016/s0960-8524(98)00085-6

Cited by 91 publications

(77 citation statements)

References 41 publications

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“…Xylose is assimilated by nonspecific hexose transporters [20], and because their affinity for glucose is many-fold higher than for xylose [17], excessive amounts of glucose competitively inhibit the transporters and prevent efficient uptake of xylose. However, coconsumption of glucose and xylose has been shown to increase the xylose uptake rate and reduce xylitol excretion, compared with xylose metabolism alone [21]. Thus, low, but non-zero, concentration of glucose enhances xylose metabolism.…”

Section: Introductionmentioning

confidence: 99%

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Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

et al. 2017

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Efficient conversion of both glucose and xylose in lignocellulosic biomass is necessary to make secondgeneration bioethanol from agricultural residues competitive with first-generation bioethanol and gasoline. Simultaneous saccharification and co-fermentation (SSCF) is a promising strategy for obtaining high ethanol yields. However, with this method, the xylose-fermenting capacity and viability of yeast tend to decline over time and restrict the xylose utilization. In this study, we examined the ethanol production from steampretreated wheat straw using an established SSCF strategy with substrate and enzyme feeding that was previously applied to steam-pretreated corn cobs. Based on our findings, we propose an alternative SSCF strategy to sustain the xylose-fermenting capacity and improve the ethanol yield. The xylose-rich hydrolyzate liquor was separated from the glucose-rich solids, and phases were co-fermented sequentially. By prefermentation of the hydrolyzate liquor followed fedbatch SSCF, xylose, and glucose conversion could be targeted in succession. Because the xylose-fermenting capacity declines over time, while glucose is still converted, it was advantageous to target xylose conversion upfront. With our strategy, an overall ethanol yield of 84% of the theoretical maximum based on both xylose and glucose was reached for a slurry with higher inhibitor concentrations, versus 92% for a slurry with lower inhibitor concentrations. Xylose utilization exceeded 90% after SSCF for both slurries. Sequential targeting of xylose and glucose conversion sustained xylose fermentation and improved xylose utilization and ethanol yield compared with fed-batch SSCF of whole slurry.

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Section: Introductionmentioning

confidence: 99%

“…The continuous release of glucose by enzymatic hydrolysis helps maintain low glucose concentrations, which kinetically favor xylose utilization in recombinant S. cerevisiae strains [21]. Typically, ethanol productivity [24] and yield are higher in SSCF than with separate hydrolysis and co-fermentation [25,26].…”

Section: Introductionmentioning

confidence: 99%

Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

et al. 2017

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“…Translated to generation time, strain TMB 3001 was stable for more than 40 generations, which is considerably longer than the four to five generations of stability reported for strain 1400 (pLNH32), which carries the same genes on a 2m-derived vector (22). Recombinant xylose-utilizing Saccharomyces strains carrying 2m-based vectors have been stably maintained in batch cultivation (22,24,27,44,48), but in continuous cultivation they tend to be unstable (27,28). The instability of strains carrying 2m-based vectors may result from genetic instability at the plasmid level, i.e., spontaneous loss of the transformed phenotype and the plasmid (19,26,28) or high frequency of recombination, resulting in cells that still carry the selectable marker but have lost the cloned gene (15,28).…”

mentioning

confidence: 99%

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1 , XYL2 , and XKS1 in Mineral Medium Chemostat Cultures

Eliasson

Christensson

Wahlbom

et al. 2000

Appl Environ Microbiol

357

269

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For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. To obtain an economically feasible industrial process for ethanol production from lignocellulose, it is necessary to ferment all sugars present with high yields and productivities (53). The commonly used Saccharomyces cerevisiae has many advantages as an ethanol producer, such as fast sugar consumption, high ethanol yield from hexoses, and high resistance to inhibitory compounds that are present in the hydrolysates. However, a major drawback is that S. cerevisiae cannot utilize the pentose sugar xylose, only its isomer xylulose. In xylose-utilizing yeasts, the conversion from xylose to xylulose is a two-step process catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH) (10), whereas bacteria perform the conversion in one step with xylose isomerase (XI) (23).Xylose fermentation by recombinant S. cerevisiae carrying heterologous XYL1 and XYL2 genes from Pichia stipitis, which encode XR and XDH, respectively, has resulted mainly in xylitol formation (24,44,48). Similarly, if xylA from Thermus thermophilus, which encodes XI, is introduced into S. cerevisiae, then only limited xylose fermentation is observed (47). Limited xylose fermentation by recombinant S. cerevisiae has been ascribed to poor xylose uptake (9, 24, 25), a cofactor imbalance generated by the discrepancy in cofactor usage by XR and XDH (8,24,49), limitations in the pentose phosphate pathway (12,24,38,48), and insufficient induction or activation of ethanologenic enzymes (5,17,20,29). When homologous XKS1, which encodes xylulokinase (XK), was overexpressed in a Saccharomyces sp. strain carrying XYL1 and XYL2, the ethanol yield and the xylose uptake rate increased under oxygenlimited conditions, but xylitol was still a major by-product (22).Although the shortcomings of xylose fermentation by recombinant S. cerevisiae have been investigated in several studies, data from anaerobic fermentations do not exist and quantitative data are sparse. Chemostat cultivations in which growth rate and concentrations of substrates and products are constant enable quantitative determinations of metabolic fluxes. Analysis o...

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“…The overexpressing stains of P. stipitis transaldolase (TAL1) gene, along with XYL2 and XYL1 implant faster growth than strains expressing XYL2 and XYL1 alone [124]. However, the plasmid burden because of the overexpression of XYL1, XYL2 and TAL1 decreased the rate of growth of the trans formant strain relative to the parental strain [125,126]. Sinorhizobium meliloti (gram-negative bacteria) defined as a model organism for investigating the plant-microbe interactions, because it participates in process of nitrogen fixation as it has symbiotic relationship with Medicago sativa and M. truncatula.…”

Section: Pentose Phosphate Pathwaymentioning

confidence: 99%

Bioethanol a Microbial Biofuel Metabolite; New Insights of Yeasts Metabolic Engineering

et al. 2018

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Scarcity of the non-renewable energy sources, global warming, environmental pollution, and raising the cost of petroleum are the motive for the development of renewable, eco-friendly fuels production with low costs. Bioethanol production is one of the promising materials that can subrogate the petroleum oil, and it is considered recently as a clean liquid fuel or a neutral carbon. Diverse microorganisms such as yeasts and bacteria are able to produce bioethanol on a large scale, which can satisfy our daily needs with cheap and applicable methods. Saccharomyces cerevisiae and Pichia stipitis are two of the pioneer yeasts in ethanol production due to their abilities to produce a high amount of ethanol. The recent focus is directed towards lignocellulosic biomass that contains 30-50% cellulose and 20-40% hemicellulose, and can be transformed into glucose and fundamentally xylose after enzymatic hydrolysis. For this purpose, a number of various approaches have been used to engineer different pathways for improving the bioethanol production with simultaneous fermentation of pentose and hexoses sugars in the yeasts. These approaches include metabolic and flux analysis, modeling and expression analysis, followed by targeted deletions or the overexpression of key genes. In this review, we highlight and discuss the current status of yeasts genetic engineering for enhancing bioethanol production, and the conditions that influence bioethanol production.

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Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1

Cited by 91 publications

References 41 publications

Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1 , XYL2 , and XKS1 in Mineral Medium Chemostat Cultures

Bioethanol a Microbial Biofuel Metabolite; New Insights of Yeasts Metabolic Engineering

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